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DISCUSSION DISCUSSION 61 Dr. Reinhold Benesch: I would like to draw attention to some work which we have done which had as its aim the de nova introduction of an -SH group into proteins through a peptide bond. Partly we had in mind the application to x-ray work since the -SH groups introduced de novo in this way could be transformed into mercury derivatives and the protein examined in this form. The compounds which we selected for this purpose are homocysteine thio- lactones, which would react with protein amino groups according to the fol- lowing scheme ~ Benesch, R. and Benesch, R. E.: I. Am. Chem. Soc. 78: 1597, 1956): S CH., ~ H., CH:3-CO-NH-CH-CO + PrNH~ ~ SH CH CH., ClI3-CO-NH-CH-CO-NH-Pr Dr. David B. Smith: Regarding the number of subunits in hemoglobin, I would like to bring to your attention some results from our laboratoryi~~~3 on horse globin. Horse globin at pH 2 and ionic strength 0.05 separates into a material whose weight-average molecular weight is 21-22,000 and whose number-average molecular weight is 17,000. These results are interpreted a. indicating four subunits with partial aggregation to give the higher weight- average molecular weight. Molecular weights were measured by osmometry, light scattering and sedimentation using Archibald's method. Incidentally, the effect of pH 2 and ionic strength 0.05 on sedimentation was checked in two ways. The sedimentation rate of ribonuclease in this medium was the same as at neutrality. The molecular weight of lysozyme by Archibald's method was about 14,000 in agreement with results obtained at neutrality and higher ionic strength. Under conditions where the molecular weight of globin has its minimum value, that is pH 2 and ionic strength 0.05, the electrophoretic pattern is at its simplest and shows two components. Extrapolation to allow for the dis- torting effect of the extreme conditions on the relative areas of the peaks indicates that the two components are present in equal amounts. We obtained small amounts of each component from the ends of the electrophoresis appa- ratus and investigated their properties separately. The faster-moving component at pH 2 and ionic strength 0.05 had a weight- average molecular weight of about 2S,000 and a number-average value of about 17,000. Any increase in pH or ionic strength resulted in association. The slower-moving component had a ~veight-average molecular weight of about 17,000 and alterations in the medium had no effect on this value. The

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62 PART I. STRUCTURE OF HEMOGLOBIN behavior of unfractionated globin is in some respects intermediate between that of these two components. We have made some investigations by Edman's methods on the amino acid sequence at the N-terminal ends of the separated components. Both compo- nents, of course, have N-terminal valine. The second amino acid residue of the faster-moving component is glutamic acid. The slower component has principally leucine in the second position; slight contamination with glutamic acid is ascribed to the difficulty of obtaining the slow component free from the faster in the descending limb of the electrophoresis cell. In conclusion, it appears that horse globin can be readily split into four subunits, all of similar molecular weight and divided equally between two types. REFEREN CES 1. Reichmann, M. E.. and Colvin, J. R.: The number of subunits in the molecule of horse hemoglobin, Can. J. Chem. 34: 411, 1956. 2. Haug, A., and Smith, D. B.: Separation, molecular weight and interactions of horse globin components, Can. l. Chem., 35: 945, 1957. 3. Smith, D. B., Haug, A., and Wilson, S.: Physical and chemical studies on horse globin components, Federation Proceedings 16: 766, 1957. Dr. V. M. Ingram: Have you any information on human globin? Dr. D. B. Smith: No. Dr. b~al~er Hughes: I would like to report an observation which may be important relative to heme-heme interaction. In searching for gentle methods or removing heme from hemoglobin, I observed that approximately half of the heme may be extracted from precipitated carbonmonoxy hemoglobin by acetone containing small amounts of pyridine and water. The resulting product appears very "native." It shows two peaks in the ultracentrifuge suggesting partial dissociation into 34,000 M.W. units. I have not been able to remove the remaining heme except by more rigorous conditions with concomitant de- naturation. Myoglobin under these conditions releases no heme. If all of the hemes are equivalent in hemoglobin, this finding must also be interpreted through heme interaction, here of a negative (repulsive) nature. Lewist has published a similar finding in the acid denaturation of carbonmonoxy hemo- globin. However, he found the removal of only the first heme to be easier than the rest. REFEREE CE 1. Lewis, U. J.: The acid cleavage of hemoglobin, J. Biol. Chem. 206: 109, 1954. Dr. M. T. Perutz: May I make a short point? I should like to remind you of a result of Kendrew and Parrishi which has some bearing on the crevice theory of iron attached in myoglobin. They prepared the 1-methyl and 4- methyl imidazole derivatives of myoglobin, which thus have large groups attached to the iron atom. They crystallized those compounds and took x-ray

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DISCUSSION 63 pictures. In taco species of myoglobin the imidazole group produced no change in the unit cell dimensions of the crystal. If there were the kind of crevice where the molecule is forced apart, as it were, through the insertion of a group like propyl isocyanide, then this ought to have the effect of making the molecule somewhat bigger and enlarging the unit cell. This, as I say, was not observed. In a third species crystallization in the usual crystal form was inhibited and replaced by another. Kendrew and Parrish conclude that the heme is most likely to be on the surface of the myoglobin molecule, the imi- dazole group finding space in the interstices between neighboring molecules in the crystal lattice. REFEREN CE 1. Kendrew, J. C. and Parrish, R. G.: Imidazole complexes of myoglobin and the position of the haem group, Nature (Lond.) 175: 206, 1955. Dr. Davidson: Dr. Perutz, would you not expect something like PC~B to change the size of a hemoglobin molecule? Dr. Edsall: You mean whether there is or is not a crevice so that merely tacking on a group as large as that to a hemoglobin will alter its dimension? Dr. Perufs: In the picture vou saw here the distance between the -SH groups was 30 Angstroms. This picture does not tell you whether the -SlI groups are on the surface or within the molecule. However, further results have now been obtained by Dr. David Green at the Royal Institution in London, which seem to show that the -SH group is about 7 Angstroms in- side the external boundary of the molecule. In other words, there must be a sort of crevice or canal where the -SH group is located, so that the PCMB does not make the molecule any bigger. I should like to mention a paper published by Drs. David Ingram. Gibson and myselt,l concerning the orientation of the neme groups. We measured the paramagnetic resonance or electron spin resonance, as it is sometimes called, of the iron atoms in single crystals of horse me/hemoglobin. We got a very beautiful and sharp anisotropic effect with the help of which it was possible to determine the angular orientation of the heme groups with a very high accuracy indeed. The accompanying figure ~ shows the hemoglobin molecule, egg shaped, 55 Angstroms wide, 55 Angstroms thick and 70 Angstroms long, lying on an axis of dyed symmetry (the lo-axis). The heme groups are arranged in two pairs, related by the dyed axis. One pair of heme normals lie in the a,b-plane of the crystal, while the other pair is tilted by 13 above and below that plane. I should like to stress that this result tells nothing about the position of the heme groups. As figure 2 shows, they can be anywhere you like, except that they must be related in pairs by the two-fold axis of symmetry. In order to find their position, we made the para-iodo-nitroso-benzene com- plex of hemoglobin, hoping that the iodines would label the iron atoms and

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64 PART I. STRUCTURE OF HEMOGLOBIN /c g ~~ ~' ~ / ~,'~ dead _ ~emoglo~ /~N ~ Fe ~ F[G. 2. Three of the many possible arrangements of the four heme groups in the hemoglobin molecule, repre- sented diagrammatically as a spheroid. In each pair of drawings the left- hand spheroid shows the molecule in projection normal to the dyed axis, and the right^hand one shows the same arrangement seen along the dyed axis. The small black points in ( c) represent the positions of the -SH groups deduced from the x-ray anal- ysis. (From Nature 178: 908, 1956.) FIG. 1.Perspective drawing of the orientation of the heme groups with respect to the crystal axes and the hemoglobin molecule. ( a ) and ( b ) show the two pairs of heme groups related to the dyed axis; (c) shows the external shape of the molecule, as determined by }3ragg and Perutz,0 drawn on a much smaller scale than the heme groups. (From Nature 178: 907, 1956.) a) \ a_ IT' Ah /~ ': ~ a a a ' 1 ~ /'' (C) in\i~ ~ THAI

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DISCUSSION help us to find their positions in an electron density map. Unfortunately, the results obtained so far have been inconclusive. REFERENCE 65 1. Ingram, D. J. E., Gibson, J. F., and Perutz, M. F.: Electron spin resonance in myoglobin and haemoglobin. Orientation of the four haem groups in haemo- globin, Nature (Lond.) 178: 905, 1956.